We have recently demonstrated that LAG-3 (CD223), a CD4 homologue that binds to MHC class II with high affinity, is a key negative regulatory T (Treg) cell protein possessing both cell intrinsic and extrinsic activity. It is required for maximal Treg function and for the control of CD4+ and CD8+ T cell homeostasis. Remarkably, ectopic expression of LAG-3 alone can confer regulatory activity. Multiple experiments have shown that a unique 'KIEELE'motif in the LAG-3 cytoplasmic domain is essential for its function. Finally, we have shown that LAG-3 is efficiently cleaved from the cell surface within the membrane proximal connecting peptide. Despite the significant progress, important questions remain that will be addressed in the following Aims: Specific Aim 1: Does LAG-3 play a key role in controlling inflammatory reactions and autoimmunity? Even though we have shown that LAG-3 is required to control T cell function, no significant inflammatory reactions or autoimmunity are seen in LAG-3 -/- mice. We hypothesized that LAG-3 may only be important during an inflammatory or autoimmune disease. Our preliminary data support this view as IBD appears to be accelerated with LAG-3 -/- T cells and autoimmunity significantly enhanced in models of T1D and EAE. Here we will determine the mechanism behind this enhanced disease by analyzing LAG-3 -/- Teff and Treg function. Specific Aim 2: How is the intrinsic regulatory activity of LAG-3 released by T cells? We have recently shown that the metalloprotease-mediated cleavage of LAG-3 significantly modulates its regulatory activity. We hypothesize that LAG-3 cleavage is required to permit normal T cell proliferation and function. This will be assessed using shRNA mediated knockdown of ADAM10 and ADAM17, T cells from ADAM10fl/fl/ADAM17fl/fl/CreERT2 mice, and analysis of a knock-in mouse expressing non-cleavable LAG-3. Specific Aim 3: What is the mechanism of intrinsic LAG-3 activity in T cells? How LAG-3 works is an important question that remains unresolved. The co-receptor dependence of LAG-3 function has led us to hypothesize that LAG-3 interferes with coreceptor (and/or TCR) function on CD4* and CDS* T cells. (A) We will assess if the disruption of phosphorylation events by LAG-3 is restricted or global using multiplexed, reverse-phase protein lysate (RPPL) microarrays. We will also determine is LAG-3 modulates p56/c/<function and downstream signaling. (B) A novel reconstitution approach will be used to generate LAG-3 -/- /CD4 -/- peripheral T cells ectopically expressing a series of co-receptor/p56/cfc mutants to assess the molecular basis for coreceptor dependence. (C) The effect of LAG-3 on CD4/TCR localization and immunological synapse formation will be determined using time-lapse confocal and TIRF microscopy. Appropriate regulation of the immune system is imperative. LAG-3 has emerged as a key negative regulatory molecule and its clear requirement for optimal regulatory T cell function warrants further study.